394nature structural biology • volume 10 number 5 • may 2003
All organisms require organometallic cofactors, such as hemes,
porphyrins and cobalamins, and must obtain them (or their pre-
cursors) from external sources. Gram-negative bacteria possess
specialized systems that bind and transport these essential mol-
ecules across two membranes (outer and inner) and the
periplasmic space for subsequent utilization within the cytosol.
Specific outer membrane transporters bind their respective sub-
strates in an energy-independent fashion1. Following the bind-
ing event, energy-dependent transport occurs across the outer
membrane. The outer membrane does not maintain an electro-
chemical gradient. The energy required for transport is obtained
by coupling the outer membrane transporter to the inner mem-
brane in a manner enabling utilization of the protonmotive force
of the inner membrane. Many of these bacterial outer membrane
transporters are coupled with the TonB protein, which is part of
an inner membrane complex that transduces energy to drive
active transport2–4. The ubiquity of TonB-dependent outer
membrane transport in Gram-negative bacteria presages this
system as a potential target for the design of new antibiotics.
TonB-dependent transporters possess a highly conserved stretch
of seven amino acid residues, the Ton box, near their
N termini5,6. Mutations in the Ton box of many outer membrane
transporters cause a loss of TonB-dependent functions with no
accompanying loss of other cellular function7–9. Thus, a key
aspect of transmembrane signaling in the TonB-dependent
transport cycle entails structural and/or dynamical changes in
the Ton box of the transporter upon substrate binding. The
X-ray crystal structures of three TonB-dependent iron-
siderophore transporters, FepA, FhuA and FecA, have been
solved10–13. Two of these, FhuA and FecA, have been solved both
in the absence and presence of bound substrate. The structure of
FepA without its siderophore substrate contains an ordered Ton
box region. Unfortunately, the FhuA and FecA structures do not
contain an ordered Ton box; thus, the structural behavior of this
functionally critical ‘coupling region’ in the presence of substrate
Numerous enzymes require cobalamin as a cofactor. In enteric
bacteria, cobalamin-containing enzymes function in amino
acid synthesis, nucleotide synthesis and metabolism14.
Methylcobalamin is the cofactor for methionine synthetase, the
enzyme that catalyzes the terminal step of methionine bio-
synthesis. Cyanocobalamin (vitamin B12), or a product related to
it, is involved in the synthesis by epoxyqueuosine reductase of the
modified base queuosine, present in the anticodon loop of some
tRNAs. Adenosylcobalamin is the cofactor for ethanolamine
ammonia lyase, which catalyzes the first step in the catabolism of
ethanolamine as a nitrogen or carbon source. All of these
various cobalamins can be obtained or produced by bacteria after
uptake of cyanocobalamin (vitamin B12). The TonB-dependent
outer membrane transporter for cobalamin in Escherichia coli is
BtuB7. As assayed in outer-membrane preparations, cyanocobal-
amin binds to BtuB with high affinity (Kd∼0.3 nM) in the pres-
ence of calcium; depletion of calcium reduces this affinity 50–100
fold15. In addition to binding and transport of cobalamins, BtuB
serves as a receptor for the E and A colicins and for bacteriophage
BF23 (refs. 16,17). The elucidation of structures of the outer
membrane transporter (BtuB, this paper), the inner membrane
ABC transporter (BtuCD)18and the periplasmic binding protein
(BtuF)19make cobalamin transport in E. coli the best structurally
characterized transport system thus far. Here, we have deter-
mined four structures of BtuB: an initial methionine-substitution
mutant used for experimental phase determination, the wild type
protein, wild type protein with bound calcium, and wild type
protein with bound calcium and cyanocobalamin (vitamin B12).
These structures, the first of an outer membrane transporter with
a substrate that is not an iron siderophore, reveal several novel
and interesting features. Among these features are a direct struc-
tural role for calcium in high-affinity substrate binding and a
conformational change in the Ton box upon substrate binding.
These structures are part of our ongoing efforts to dissect the
molecular basis of TonB-dependent outer membrane transport.
Substrate-induced transmembrane signaling in
the cobalamin transporter BtuB
David P. Chimento1,2, Arun K. Mohanty1,3, Robert J. Kadner2and Michael C. Wiener3
Published online 24 March 2003; doi:10.1038/nsb914
The outer membranes of Gram-negative bacteria possess transport proteins essential for uptake of scarce nutrients.
In TonB-dependent transporters, a conserved sequence of seven residues, the Ton box, faces the periplasm and
interacts with the inner membrane TonB protein to energize an active transport cycle. A critical mechanistic step is
the structural change in the Ton box of the transporter upon substrate binding; this essential transmembrane
signaling event increases the affinity of the transporter for TonB and enables active transport to proceed. We have
solved crystal structures of BtuB, the outer membrane cobalamin transporter from Escherichia coli, in the absence
and presence of cyanocobalamin (vitamin B12). In these structures, the Ton box is ordered and undergoes a
conformational change in the presence of bound substrate. Calcium has been implicated as a necessary factor for
the high-affinity binding (Kd∼0.3 nM) of cyanocobalamin to BtuB. We observe two bound calcium ions that order
three extracellular loops of BtuB, thus providing a direct (and unusual) structural role for calcium.
1These authors contributed equally to the work. 2Department of Microbiology and 3Department of Molecular Physiology and Biological Physics, University of Virginia,
Charlottesville, Virginia 22908, USA.
Correspondence should be addressed to M.C.W. e-mail: firstname.lastname@example.org
© 2003 Nature Publishing Group http://www.nature.com/naturestructuralbiology
nature structural biology • volume 10 number 5 • may 2003401
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